The primary goal of this research project is to reveal and analyze genes involved in the regulation of neurotoxin (BoNT) production in Clostridium botulinum type A strain 62A. Nontoxigenic (tox-) mutants and mutants with significantly decreased BoNT production, as well as mutants affected in synthesis of nontoxic components of the type A toxin complex, will be isolated after insertion mutagenesis with the conjugative transposon Tn916. Mutants affected in toxinogenesis will be identified by screening colonies by an immunoblot assay and by ELISA in microtiter plates. Detailed Southern hybridization analyses of the parent and mutant strains will be performed to locate regulatory genes on the clostridial chromosome, and to reveal which genes are regulated. The clostridial chromosomal fragments containing regulatory regions will be cloned, their sequence determined and analyzed. The role of the virulence genes will be determined by complementation of the mutant strain with the wild-type allele containing the region of interest isolated from the parent strain. Transcription and translation patterns of BoNT and nontoxic components of the toxin complex will be analyzed in C. botulinum 62A by Northern analyses, by Western blots, and by ELISA. Knowledge gained in these studies will have practical value in the development of improved botulinum toxin complexes as pharmaceuticals and for understanding the capacity of C. botulinum to cause foodborne disease and human and animal infections.